Resources

Over the last fifteen years the Danforth lab has developed protocols and primers for molecular systematics of bees. While the details of most of these protocols are available in published papers, we present below a summary of protocols, background information on genes, primer sequences, gene maps, and related literature.

If you find this information useful, please cite it in your published papers as:

A. Ribosomal genes

Insect molecular systematists interested in reconstructing deeper (i.e., Mesozoic and older) divergences are presented with a choice of using nuclear ribosomal genes or nuclear single-copy, protein-coding genes. Mitochondrial genes are generally considered too rapidly evolving for these deep divergences (Moriyama & Powell 1997) and show substitution patterns that are problematic for reconstructing ancient divergences (Lin & Danforth 2004). We do not recommend using mitochondrial genes for higher-level studies in bees.

1. 28S D2-D7 region

While we prefer single-copy, protein-coding genes for higher-level bee phylogeny, regions of the 28S D2-D3 region have been used previously in bees (Cameron & Mardulyn 2001). We have successfully amplified and sequenced the same region used by Cameron & Mardulyn (2001) for a broad survey of ST and LT bees. Our data set and the one by Cameron & Mardulyn (2001) were generated using primers D2-3665F (Bel28S) and D3-4283R (Mar28Srev) (see primer lists and maps in the attached pdf files below). The resulting alignment has some highly conserved regions, but several expansion regions (loops) make the alignment ambiguous. Virtually all the primer pairs listed in the attached pdf file amplify some or all bees tested. The complete honey bee 28S sequence is available on GenBank as accession number AF181590.

Litman, J.R., B.N. Danforth, C.D. Eardley, & C.J. Praz (2011). Why do leafcutter bees cut leaves? New insights into the early evolution of bees. Proc. Royal Society of London (B)

2. 18S gene

18S is a commonly used ribosomal gene for studies of higher-level (ordinal level) relationships in insects (Caterino et al. 2000). Chalwatzis et al. (1996), for example, were able to use 18S to help determine the relationships between all the major groups of the Holometabola, though they noted that 18S could be problematic for phylogenetic analysis because of variable G+C content in different regions of the gene. Nonetheless, Caterino et al. (2000) asserted that “given satisfactory results from the 18S data and a substantial existing database, it seems advisable to concentrate future efforts in this region”. 18S has been widely used in recent insect ordinal-level studies (Whiting et al. 1997, Wheeler et al. 2001, Whiting 2002, Kjer 2004).

The only previous study using 18S in bee phylogeny is that of Sheppard & McPheron (1991). In this study 18S was used to resolve relationships within the corbiculate Apidae. However, their alignment included just 237 nucleotide sites, seven of which were variable. We have chosen to use 18S for our studies of bee family level phylogeny because the gene is easy to amplify and may help provide resolution at the base of the tree. We are still in the process of assessing the utility of this gene for higher-level studies in bees.

Below is a figure, scanned from Caterino et al. (2000), which shows a map of ribosomal DNA and datasets including 18S sequences that were published prior to March 15, 1999.

B. Nuclear protein-coding genes

Slowly-evolving, nuclear, protein-coding genes have recently become available for phylogenetic analysis in many groups of insects. These genes have a number of advantages over ribosomal genes. Most obviously, they are easily alignable. Many of these genes have been demonstrated to be capable of recovering Creteaceous age divergences in insects (Friedlander et al. 1992, 1994, 1996, 1997, 2000; Moulton & Wiegmann 2004; Wiegmann et al. 2000).

1. Elongation factor-1alpha

Elongation factor-1α (EF-1α) is a nuclear gene that encodes a protein involved in the GTP-dependent binding of charged tRNAs to the acceptor site of the ribosome during translation (Maroni 1993, pp. 126-134). EF-1α has proven to be a useful gene for studies of higher-level phylogenetic relationships, especially in insects (Cho et al. 1995; Friedlander et al. 1998; Mitchell et al. 1997, 2000; Moulton 2000; Regier et al. 2000; Caterino et al. 2000). Amino acid sequences of EF-1α have been used to resolve relationships among arthropod classes (Regier & Shultz 1997, 1998; Shultz & Regier 2000), worm phyla (Kojima et al. 1993; McHugh 1997, 2000; Eeckhaut et. al 2000), and among early eukaryotes (Hasegawa, et al. 1993; Kamaishi, et al. 1996). EF-1α occurs as two copies in bees (Danforth & Ji 1998), ants (T. Schultz, pers. comm.), and flies (Hovemann et al. 1988). We have developed primers that specifically amplify the F1 or the F2 copy in bees (Danforth et al. 1999, Danforth & Ji 2001, Sipes & Wolf 2001). Paralogs can be easily distinguished based on the positions of introns, which differ among copies (see attached pdf file with a map of both the F1 and F2 copies). We typically sequence nearly the entire F2 copy (roughly 1600 bp, with three introns) for most of our studies (primers for this paralog are listed below in the pdf file). Introns are difficult to align unambiguously at the family level and, while some conserved non-coding regions may be retained, most intron sequence may need to be excluded from the analysis.

A recent study (Brady & Danforth 2004) demonstrated that there is intron presence/absence variation in the F1 copy of EF-1α. All members of the family Colletidae that we sampled had an intron that was lacking in all other bees, sphecid wasps, and ants that we sampled. We interpreted this as a uniquely derived intron that supports the monophyly of the colletid bees and excludes the Stenotritidae from the Colletidae.

We have typically used the F2 copy of EF-1α (e.g., Danforth et al. 2004) because our primers (see pdf files below) can amplify a large and continuous fragment of approximately 1600 bp which includes two introns. However, a number of bee studies have used the F1 copy (e.g., Leys et al. 2002, Bull et al. 2003; Schwarz et al. 2003, 2004). The fragment analyzed in these studies is approximately 500 bp in length and includes only exon sequence (except in colletids in which there is an intron; Brady & Danforth 2004). We prefer the F2 copy because of its ease of amplification and because there is a substantial amount of data already available for this copy.

The honey bee (Apis mellifera) F2 copy is available on Genbank as accession number AF015267 (Danforth & Ji 1998). The honey bee (Apis mellifera) F1 copy is available on Genbank as accession number X52884 (Walldorf & Hovemann 1990).

2. LW rhodopsin

The bee long-wavelength rhodopsin is one of a family of four rhodopsin genes in bees: two long wavelength (green) paralogs, one blue, and one UV rhodopsin (Towson et al. 1998, Chang et al. 1996, Briscoe 2002, Spaethe & Briscoe 2004). Rhodopsins are G-protein-coupled receptor proteins that perform the first steps in visual transduction in most organisms. Long-wavelength rhodopsins have been characterized and sequenced in ants (Popp et al. 1996), mantids (Towner & Gärtner 1994), butterflies (Briscoe 1999, 2000, 2001) and flies (Drosophila, Huber et al. 1997). The long-wavelength rhodopsin (hereafter LW opsin) has been used for phylogenetic analysis of bees (Mardulyn & Cameron 1999, Cameron & Mardulyn 2001, Ascher et al. 2001, Cameron & Williams 2003), Heliconius butterflies (Hsu et al. 2001), cynipid wasps (Rokas et al. 2002, Nylander et al. 2004), and aphids (Ortiz‑Rivas et al. 2003). Interestingly, Spaethe & Briscoe (2004) recently described a new LW opsin paralog (they called it LW Rh2 to distinquish it from the previously described LW Rh1). This paralog differs from LW Rh1 in the location of several introns and is substantially different at the nucleotide level. They list primers that amplify this paralog in diverse genera of bees including Bombus, Diadasia and Osmia. This copy has not been used in phylogenetic analysis of any insects, but could provide an additional data set.

Previous phylogenetic analyses of bees (Mardulyn & Cameron 1999, Cameron & Mardulyn 2001, Ascher et al. 2001, Cameron & Williams 2003) used opsin primers LWRhFor, and LWRhRev, which produce an approximately 700 bp PCR product consisting of three exons and two introns from the LW Rh1 paralog (primers are listed in the attached pdf file below). We expanded the opsin data set beyond the range of previous studies using 3′-RACE, as described below. Poly-A mRNAs were isolated from heads of recently collected, frozen bees (Agapostemon virescens, Andrena carlini, Augochlorella striata, Lasioglossum leucozonium, Colletes inaequalis, Halictus ligatus, and Lasioglossum (Dialictus) zephyrum; all collected in Ithaca, NY) using Oligotex Direct mRNA Micro Kit (Qiagen, Chatsworth, CA). The mRNA were then used for reverse-transcription to generate cDNA. PCR of cDNA fragments using opsin-specific primers and poly-T reverse primers were used to generate PCR products, which were then ligated into a pGEM vector (Promega, Madison, WI), and transformed into DH5-α library efficiency competent cells (Gibco, Grand Island, NY). The transformed cells were plated out on LB medium. Plasmids containing opsin inserts were isolated and sequenced using an ABI 373A automated sequencer. We aligned the sequences obtained with the three different opsin copies in Apis (Chang et. al 1996) to confirm that the cloned fragments matched the LW gene family. All sequences spanned the 3′ end of the gene and all included the stop codon plus the 3′ non-coding region. Based on these sequences we developed two reverse primers (Opsin Rev4, Opsin Rev4a; see pdf file below) to amplify the 3′ end of the gene (including one intron).

Our opsin data set spans positions 434 to 1146 of the coding region in the Apis mellifera LW opsin paralog (Chang et al. 1996), and includes four exons and three introns (see attached pdf file with the map of the gene). The region we sequenced spans transmembrane regions III to VII plus the intracellular loop downstream of transmembrane region VII. The locations of introns coincide with the junction of transmembrane and extra-membrane regions, suggesting that the introns flank functional domains of the protein.

3. Wingless (Wnt1 gene family)

The wnt family of protein coding genes are involved in early embryogenesis in insects and vertebrates (e.g. Uzvölgyi et al. 1988; Rijsewijk et al. 1987). At least a dozen subfamilies have been identified in this large and variable gene family (Sidow 1992, Schubert et al. 2000, Prud’homme et al. 2002). Of these subfamilies, wnt-1 has been shown to be phylogenetically informative at a variety of levels, including among phyla of metazoan animals (Schubert et al. 2000), among Hawaiian carabid beetles (Cryan et al. 2001), and among and within Lepidopteran families (Brower & DeSalle 1998, Brower & Egan 1997, Brower 2000, Campbell et al. 2000, Wahlberg et al. 2003). A recent study of phylogenetic relationships within the stalk-eyed flies (family Diopsidae) concluded that, of the six genes analyzed, wingless proved to be the most useful (in terms of congruence, data decisiveness, and bootstrap support; Baker et al. 2001).

In a preliminary evaluation of the wingless gene for relationships among the bee families, subfamilies and tribes, we used primers developed for Lepidoptera (wg1a and wg2a; Brower & DeSalle 1998). These primers non‑specifically amplified two to three wingless paralogs in most bee and spheciform taxa. Two paralogs differed in size and could be separated on low melting point agarose gels. When paralogs were similar in size we used PCR product cloning (T/A cloning) to isolate and sequence alternative paralogs. We characterized a total of three wingless paralogs in bees that could be separated unambiguously based on small or large indel mutations. The three paralogous copies in bees clustered unambiguously into three different wingless gene families. Our most common paralog is in the wnt-1 family. Based on these wnt-1 sequences, we designed a new forward PCR primer (bee wg For) that specifically amplifies 450 bp of the wnt-1 paralog in a variety of ST bee families and sphecid outgroups. Preliminary phylogenetic analysis of 65 halictid wnt-1 sequences recovered subfamilies, tribes and genera that have been well supported by morphology and other single-copy nuclear genes (Danforth et al. 2004). Sedonia Sipes (Southern Illinois University, Carbondale) developed additional wingless primers using 5′ RACE to amplify and sequence an upstream portion of the gene in a broad sample of ST bees. Eduardo Almeida (Almeida & Danforth 2009) developed additional primers for amplifying an even larger fragment of the wingless gene. These primers appear to work well across a broad survey of bee families. Currently available primers are listed in the pdf file below.

The Drosophilawingless sequence is available on GenBank as accession number J03650 (Uzvölgyi et al. 1988).

4. Arginine kinase

Arginine kinase is an important metabolic enzyme that is highly expressed in the brain, antennae, and compound eyes in bees (Kucharski & Maleszka 1998). Complete sequences are available for the honey bee (Apis mellifera; AF023619), fruit-fly (Drosophila melanogaster), and migratory locust (Schistocerca gregaria). Primers developed for phylogenetic studies of bumble bees (Kawakita et al. 2003) work on a diversity of short-tongued bees, including members of the the family Colletidae. We are in the process of expanding this approximately 500 bp data set using newly developed primers from the 3′ end of the gene. We are currently able to amplify and sequence an approximately 1000 bp fragment of this gene, including two introns. This gene appears to evolve at roughly the same rate as LW rhodopsin. Primers and a map of the gene are provided in the pdf files below.

5. PEPCK

Phosphoenolpyruvate carboxykinase (PEPCK) is a single copy nuclear gene involved in gluconeogenesis in insects and other organisms (Friedlander et al. 1992, 1994). The gene has been demonstrated to be useful for recovering Cretaceous-age divergences in Lepidoptera alone (Friedlander et al. 1996) or in combination with other genes (Wiegmann et al. 2000), and has been used to recover relationships in carabid beetles (Sota & Vogler 2001) and simuliid flies (Moulton 2000). Recently, PEPCK was used in a phylogenetic analysis of the LT bee tribe Xylocopini (in the apid subfamily Xylocopinae; Leys 2000; Leys et al. 2000, 2002). Results based on a roughly 1 kb fragment of PEPCK, including three exons and two introns, indicates that the gene is useful for recovering Tertiary or late Cretaceous age divergences in bees (Leys 2000, Leys et al. 2002). Most nodes in an equal weights parsimony analysis of PEPCK were supported by bootstrap values >60% and relationships implied by the PEPCK data were congruent with a recent morphological study (Minckley 1998) and data from EF-1α (Leys et al. 2002).

6. CAD (rudimentary)

CAD, also known as a gene called rudimentary, is a nuclear, protein-coding gene that is involved in pyrimidine biosynthesis. (The gene was originally named rudimentary because mutant forms of the gene caused deformed wings in Drosophila.) CAD is a single copy gene and, in Drosophila and Anopheles, it is found on the X chromosome. Full-length CAD sequences are available for Drosophila melanogaster (GenBank accession number AE003503) and Anopheles gambiae (GenBank accession number EAA06526). In Drosophila and other insects, CAD consists of four distinct loci that catalyze the first steps in the de novo pyrimidine biosynthetic pathway: carbamoylphosphate synthetase (CPS), dihydroorotase (DHO), aspartate transcarbamylase (ATC), and glutamate aminotransferase (GAT) (Moulton & Wiegmann 2004, Freund & Jarry 1987). In insects and vertebrates the largest of the CAD domains is the carbamoylphosphate synthase (CPS) domain (Kim et al. 1992). CPS consists of approximately 4 kb of coding sequence, and provided robust support for higher-level, Cretaceous-age divergences in Diptera (Moulton & Wiegmann 2004).

Using the recently published sequence of the honey bee genome, we located the CAD homolog by blasting with fly CAD sequences. We assembled a fragment of CAD that matches very well the large data set assembled for flies by Moulton & Wiegmann (2004). Using this alignment we developed primers for amplifying and sequencing an approximately 3000 bp fragment of CAD in bees (see pdf file below). Based on preliminary tests, these primers work well on a broad survey of ST and LT bees.

Interestingly, CAD contains several small introns (usually 50-60 bp) that vary in presence and absence among taxa. Intron presence/absence could be coded as a character when introns vary in position among taxa (Brady & Danforth 2004). CAD seems to have a slight A/T bias (~60%) in Diptera (Moulton & Wiegmann 2004).

7. RNA polymerase II

Pol II refers to the protein coding gene which codes for the two largest subunits of the RNA Polymerase II enzyme. RNA polymerase II is involved in the synthesis of pre-mRNA (Shultz & Regier 2000, Liu et al. 1999). It is a single copy gene in fungi, though paralogous copies have been discovered in plants and trypanosomes. It is not entirely clear whether Pol II is a single copy or multiple copy gene in insects, though our lab has not encountered any evidence of paralogous copies in bees. Pol II is easy to amplify and align and contains several highly conserved regions which simplifies primer design (Liu et al. 1999).

The three-dimensional structure of Pol II has been described in Cramer et al. (2001), and there are already a substantial number of sequences available for this gene (Schultz & Regier 2000). The fragment that we have been able to amplify comes from the RPB2 gene, which encodes the largest of the Pol II subunits, and is involved in the catalyzation of the elongation reaction in mRNA transcription (Liu et al. 1999). The function of the RNA polymerases are common among all eukaryotic organisms (Sidow & Thomas 1994). Shultz & Regier (2000) demonstrated the utility of Pol II in resolving higher-level arthropod phylogeny, and Liu et al. (1999) showed that intron insertion/deletions occur within the Ascomycetes.

Using the recently published sequence of the honey bee genome, we located the Pol II homolog by blasting with partial insect Pol II sequences (from Shultz & Regier 2000) . Using this alignment we developed primers for amplifying and sequencing an approximately 2000 bp fragment of Pol II gene in bees (see pdf file below). Based on preliminary tests, these primers work on a broad survey of bees.

Pol II is probably the slowest single copy, protein-coding, nuclear gene that we have surveyed. Pol II evolves at approximately four times the rate of nuclear 18S ribosomal sequences (Sidow & Thomas 1994) but considerably slower than the other protein-coding genes described above. It is also more phylogenetically informative, recovering more lineages in Metazoa than 18S alone (Sidow & Thomas 1994). We are currently assessing the utility of Pol II for family level bee phylogeny using the primers listed on the attached pdf file. We have found that the region spanned by our primers contains no introns in any bees or wasps analyzed.

8. Elongation factor 2

Elongation Factor 2 (EF2), or Ca2+/calmodulin-dependent protein kinase III, is a conserved gene that encodes for the calcium and calmodulin-dependent, GTP-binding Elongation Factor 2 kinase. EF2 is not a part of the main Ser-Thr-Tyr protein kinase family (Ryazanov et al. 1997) and is a single copy gene in insects. EF2 is a cytoplasmic protein that functions in moving the ribosome down the mRNA during translation (Ryazanov et al. 1997). In eukaryotes, EF2 provides the ability to regulate mRNA translation through its numerous phosphorylation sites. When phosphorylated, EF2 is incapable of binding ribosomes, rendering the protein inactive (Browne & Proud 2004).

EF2 has been used in phylogenetic studies of fungi (Kullnig-Gradinger et al. 2002) and arthropods (Pietrantonio et al. 2002; Regier & Schultz 2001). EF2 was able to recover some clades in arthropods with strong bootstrap support and was considered by Regier & Schultz (2001) to be a promising gene equal to EF-1α and PolII for higher-level arthropod studies.

We were able to identify the EF2 gene in Apis using the recently published honey bee genome. This gene is highly conserved when compared with other insects sequences (generated by Regier & Schultz 2001). However, in bees there is a relatively large number of short introns (9 in the region we analyzed in Apis) that may make it difficult to amplify a single large fragment of exon. EF2 has similar rates of nonsynonymous substitution and pairwise differences as EF-1α, and contains approximately eight indel regions which differ among taxa, and which can be used as characters for phylogenetic analysis (Regier & Schultz 2001).

9. Abdominal-A

Abdominal A (abd-A) encodes a product with specific RNA polymerase II transcription factor activity involved in oenocyte development which is localized to the nucleus. It is expressed in the Drosophila embryo and larva (Flybase web site http://flybase.bio.indiana.edu/). Its amino acid sequence contains a homeobox domain and a ‘Homeobox’ antennapedia‑type domain. It interacts genetically with a number of developmental genes including Ubx , Abd‑B , Dfd , and lab. The gene consists of three exons: (1) a 5′ highly conserved 750 bp exon, (2) a small (36 bp), highly conserved second exon, and (3) a third exon containing a homeobox domain. Similarity between the homeobox domain of abd-A and other homeobox genes (such as Ultrabithorax and Antennapedia) makes this third exon a poor choice for phylogenetic studies. DeMenten et al. (2003, Fig. 2) provide a map of the gene. Complete sequences of the abd-A gene are available on Genbank for Tribolium castaneum [Genbank number AF017415], Drosophila melanogaster[Genbank number X54453], Anopheles gambiae [Genbank number AF080566], and Myrmica rubra [Genbank number AF332515] (Niculita et al. 2001).

Abd-A has been used in phylogenetic studies of ants (Astruc et al. 2004, Ward & Downie 2004) as well as in studies of sex determination (DeMenten et al. 2003). Primers developed initially by DeMenten et al. (2003) span an approximately 700 bp region in the first exon of the abd-A gene (with no introns). Primers were later improved by Ward & Downie (2004) in a phylogenetic study of ants. The utility of this gene relative to the others used by Ward & Downie (2004) was not discussed, although the gene appears to be a highly conserved one (sequence divergence ranged from 0.0% to 18%). The gene appears to be a reasonable one for higher-level bee phylogeny. Apis melliferaabd-A sequence is available under Genbank accession number AY703685 (Ward & Downie 2004). Using this fragment to search the honeybee genome, we were able to locate a complete abd-A fragment for the honeybee. Primers developed for ants and bees (Ward & Downie 2004), a map of the gene (from DeMenten et al. 2003), and an alignment of Apis and Myrmicaabd-A are provided in the files listed below.

10. Na/K ATPase

Sodium‑potassium ATPase (or NaK) is an ion pump responsible for establishing an electrochemical gradient across the cell membrane (Reeves & Yamanaka 1993). It is a P‑type (or E1E2) ATPase ion co‑transporter, meaning that it works via the phosphorylation of a covalent intermediate state (Emery et al. 1998). Like other P‑type ATPases, NaK is sensitive to vanadate, alternates between an E1 and E2 conformational state, and has a large highly conserved polypeptide chain usually greater than 100 kDa, usually designated the alpha‑subunit (Fagan & Saier Jr. 1993). Found in the plasma membrane (Emery et al. 1998), it pumps 3 Na+ ions out of the cell and 2 K+ ions into the cell (Emery et al. 1998), and the subsequent electrochemical gradient that is established powers several other vital transport processes and cell signaling functions across the cell membrane (Reeves & Yamanaka 1993, Emery et al. 1998). This gene was identified as a promising candidate for insect phylogeny by Friedlander et al. (1992) but has not been used in previous studies of insect phylogenetics.

NaK is a heterodimer composed of the catalytically active alpha‑subunit (containing approximately 1000 amino acids residues) and a glycosylated beta‑subunit, the function of which is unknown (Emery et al. 1998, Reeves & Yamanaka 1993). The highly conserved alpha‑subunit has been historically divided into three segments: Segment 1 consists of the N‑terminus, four putative transmembrane helices, and some cytoplasmic and extracellular loops, including the ouabain‑binding site. Segment 2 includes the large cytoplasmic region that contains the phosphorylation domain and the ATP binding site. Segment 3 contains the remaining C‑terminal region (Fagan & Saier Jr. 1993).

In invertebrates, NaK is a single copy nuclear protein‑coding gene that can undergo post‑transcriptional modifications to produce different transcripts (Reeves & Yamanaka 1993). Vertebrates, however, have evolved multiple copies of both the alpha and beta subunits, which allow for greater tissue specificity (Emery et al. 1998).

The NaK pump is easily disabled by the addition of ouabain, a cardiac glycoside (Labeyrie & Dobler 2004) and several studies have been carried out looking at the evolution of ouabain resistance in species of insects that feed on milkweed plants (Labeyrie & Dobler 2004, Holzinger & Wink 1996, Moore & Scudder 1986, Vaughan & Jungreis 1977). Tissue‑specific expression of NaK ATPase varies widely among species. Blood‑feeding insects, for example, will express NaK in higher concentration within their midgut, where it acts to remove Na+ and water from the intestinal lumen (Emery et al. 1998).

Our primers were developed by examining an alignment of the ouabain‑binding site of beetles presented in Labeyrie & Dobler (2004), and by blasting the subsequent consensus against the Apis whole genome. The fragment amplified by our primers is an intronless region, about 1.5kb long, that stretches across the extracellular ouabain‑binding site of the alpha‑subunit. It is found near the N‑terminus of Segment 1 of the NaK alpha‑subunit, and studies of mutant NaKs indicate that the ouabain‑binding site is on the first extracellular loop, between the first and second transmembrane helices (Fagan & Saier Jr. 1993).

Complete sequences of the Na/K ATPase gene are available on Genbank for Drosophila melanogaster [Genbank number AF044974], Ctenocephalides felis [Genbank number S66043] (Labeyrie & Dobler 2004).

Minckley, R.L. (1998). A cladistic analysis and classification of the subgenera and genera of the large carpenter bees, tribe Xylocopini (Hymenoptera: Apidae). Scientific Papers, University of Kansas Natural History Museum 9:1-47.

Schubert, M., L.Z. Holland, N.D. Holland, & D.K. Jacobs (2000). A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate Amphioxus. Mol. Biol. Evol. 17:1896-1903.